1. Field of the Invention
This invention relates to an assay kit and method for detecting the presence and extent of substances capable of inhibiting de novo biomolecular synthesis, such as toxicants, and antibiotics.
2. Description of Background and Relevant Information
Conventional toxicity bioassays using animals or animal cells are time consuming and expensive to perform. Of short term bioassays which are presently known, bacterial tests are the most suitable for screening chemical toxicity because they are easy to perform, reproducible and not expensive. Existing microbial tests measure growth inhibition, oxygen uptake, luminescence, colony-forming ability, ATP content and enzymatic activity.
Field-oriented assays are needed which are quick, sensitive and simple, for detection of toxicants in water, chemicals, pharmaceuticals, cosmetics, foodstuffs and food additives. By way of example, field testing is essential in antibiotic testing in milk, and in sera and urine.
A number of bacterial toxicity screening systems in use include a bioluminescence test (Microtox test of Beckman) based on the principle of light reduction upon exposure to toxicants (QURESHI et al., Article entitled Evaluation and Refinement of the Microtox Test for use in Toxicity Screening in "Toxicity Screening using Procedures Bacterial Systems", edited by Liu et al., National Water Research Institute, Burlington, Ontario, Canada 1984, pp. 1-2; the Spirillum volutans test for use in toxicity screening (GOATCHER et al., Article entitled Evaluation and Refinement of the Sprillum Volutans Test for Use in Toxicity Screening, in Toxicity Screening, Supra., pp. 89-108, 1984 and the tetrazolium reduction assay (KOOPMAN et al., Article entitled Validity of Tetrazolium Reduction Assays for Assessing Toxic Inhibition of Filamentous Bacteria in Activated Sludge, in Toxicity Screening, Supra., pp. 147-162, 1984.
The first system is quite sensitive but requires expensive instrumentation. The other methods are complicated to perform or lack the proper sensitivity required for toxicant analysis in water and foodstuffs. The literature discloses additional techniques in which relatively rapid and sensitive assaying is possible based upon observation of protein synthesis inhibition.
By way of example, streptomycin has been found to inhibit de novo protein synthesis, and to only partially inhibit ongoing (translational) protein synthesis (ZIERBUT et al. Eur. J. Biochem. 98: 577-583, 1979; DAVIS et al., Article entitled: "Complex Interaction of Antibiotics with the Ribosomes" in "Ribosomes" edited by Nomura et al., Cold Spring Harbor Laboratory, pp. 755-762, 1974; CHOI et al., Biochem. Biophysics Res. Comm. 94: 755-762 1980 ARTMAN et al., Antimicrob. Agents Chemother. 7: 449-455, 1972. Utilizing the inhibition or protein synthesis, it is possible to assay for the presence of a toxicant or the like. However, such a technique has been possible only with toxicants and the like which are fast acting, and which rapidly inhibit de novo protein synthesis. Where a toxicant is slow acting, it could not be assayed using this technique because there is insufficient inhibition before translational synthesis begins.
It would, therefore, be desirable if such techniques could be improved in a way which would also permit the assaying of substances which inhibit de novo protein synthesis, but which are slower acting.
There is thus a demand for a rapid, field-oriented kit with high sensitivity, whose procedures are simple to perform and which gives visibly detectable results, even with substances which themselves only relatively slowly inhibit de novo protein synthesis.